This Phase I SBIR proposal will advance the characterization of TTL-3, a highly potent derivative of a naturally occurring and orally available small molecule that inhibits tumor necrosis factor-alpha (TNF-alpha) synthesis. TTL-3 was designed and synthesized, and its potency will be assessed in assays to be optimized in-house. These assays include in vitro measurements of dose and time responses of cytokine gene expression by lipopolysaccharide (LPS)-stimulated human and murine lymphoid cells treated with TTL-3. The secretion of the pro-inflammatory cytokines, TNF-alpha, interleukin-1 (IL-1), and interleukin-6 (IL-6) and the anit-inflammatory cytokine, interleukin-10 (IL-10), will be monitored by ELISA. The safety and oral availability of TTL-3 will be addressed in a murine model of escalating dose tolerance. TTL-3 dose and time response and efficacy will be investigated in an acute TNF-alpha-induced murine LPS mediated injury model and a chronic model of collagen-induced (CIA) arthritis in mice. These studies will address TTL-3 potency and mechanism for TNF-alpha inhibition and will provide an important basis for further assessment of therapeutic potential in the attached Phase II SBIR studies and in a Phase I/II clinical trial for rheumatoid arthritis. PROPOSED COMMERCIAL APPLICATION: The clinical efficacy of injectable drugs such as Enbrel and Remicade in RA and IBD has proven the importance of targeting TNF-alpha. However the multiple side effects of these inhibitors suggests certain advantages of an oral small molecule TNF-alpha synthesis inhibitor such as TTL-3 over other approaches: Oral dosing with rapid absorption for greater patient acceptability; Specific activity for pro-inflammatory cytokine, TNF-alpha; Pharmacology suggests no long-term immunosuppression; Non-immunogenic, small molecule with easier manufacturing and lower cost; Potential use in combined therapy with other anti-inflammatory therapies. The understanding of the clinical activities of TNF-alpha inhibitors has simplified our ability to develop TTL-3. The potential use of TTL-3 alone or in combination with other anti-inflammatory therapies opens new clinical and marketing opportunities.